BackgroundColorectal cancer (CRC) is a common cancer worldwide. The main cause of death in CRC includes tumor progression and metastasis. At molecular level, these processes may be triggered by epithelial-mesenchymal transition (EMT) and necessitates specific alterations in cell metabolism. Although several EMT-related metabolic changes have been described in CRC, the mechanism is still poorly understood.ResultsUsing CrossHub software, we analyzed RNA-Seq expression profile data of CRC derived from The Cancer Genome Atlas (TCGA) project. Correlation analysis between the change in the expression of genes involved in glycolysis and EMT was performed. We obtained the set of genes with significant correlation coefficients, which included 21 EMT-related genes and a single glycolytic gene, HK3. The mRNA level of these genes was measured in 78 paired colorectal cancer samples by quantitative polymerase chain reaction (qPCR). Upregulation of HK3 and deregulation of 11 genes (COL1A1, TWIST1, NFATC1, GLIPR2, SFPR1, FLNA, GREM1, SFRP2, ZEB2, SPP1, and RARRES1) involved in EMT were found. The results of correlation study showed that the expression of HK3 demonstrated a strong correlation with 7 of the 21 examined genes (ZEB2, GREM1, TGFB3, TGFB1, SNAI2, TWIST1, and COL1A1) in CRC.ConclusionsUpregulation of HK3 is associated with EMT in CRC and may be a crucial metabolic adaptation for rapid proliferation, survival, and metastases of CRC cells.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4477-4) contains supplementary material, which is available to authorized users.
BackgroundCarotid body tumor (CBT) is a form of head and neck paragangliomas (HNPGLs) arising at the bifurcation of carotid arteries. Paragangliomas are commonly associated with germline and somatic mutations involving at least one of more than thirty causative genes. However, the specific functionality of a number of these genes involved in the formation of paragangliomas has not yet been fully investigated.MethodsExome library preparation was carried out using Nextera® Rapid Capture Exome Kit (Illumina, USA). Sequencing was performed on NextSeq 500 System (Illumina).ResultsExome analysis of 52 CBTs revealed potential driver mutations (PDMs) in 21 genes: ARNT, BAP1, BRAF, BRCA1, BRCA2, CDKN2A, CSDE1, FGFR3, IDH1, KIF1B, KMT2D, MEN1, RET, SDHA, SDHB, SDHC, SDHD, SETD2, TP53BP1, TP53BP2, and TP53I13. In many samples, more than one PDM was identified. There are also 41% of samples in which we did not identify any PDM; in these cases, the formation of CBT was probably caused by the cumulative effect of several not highly pathogenic mutations. Estimation of average mutation load demonstrated 6–8 mutations per megabase (Mb). Genes with the highest mutation rate were identified.ConclusionsExome analysis of 52 CBTs for the first time revealed the average mutation load for these tumors and also identified potential driver mutations as well as their frequencies and co-occurrence with the other PDMs.Electronic supplementary materialThe online version of this article (10.1186/s12920-018-0327-0) contains supplementary material, which is available to authorized users.
Fragmentation of DNA is the very important first step in preparing nucleic acids for next-generation sequencing. Here we report a novel Fragmentation Through Polymerization (FTP) technique, which is a simple, robust, and low-cost enzymatic method of fragmentation. This method generates double-stranded DNA fragments that are suitable for direct use in NGS library construction and allows the elimination of the additional step of reparation of DNA ends.
As public health systems are being modernized across the world, conventional medicine is undergoing a serious transformation and new medical models are emerging based on personalized, predictive, participatory, precision, mobile, and digital approaches. So far, there is no consensus in the literature and the medical community about the goals, objectives and applications of these models, particularly precision medicine, which is sometimes perceived as merely a fancier term for personalized medicine. The role of laboratory diagnostics in precision medicine is also a matter of intense debate. This review analyzes the currently available information about precision medicine and gives examples of how 5P approaches can be used in clinical practice.
21Fragmentation of DNA is the first and very important step in preparing nucleic acids for NGS. 22 Here we report a novel Fragmentation Through Polymerization (FTP) technique, which is simple, 23 robust and low-cost enzymatic method of fragmentation. This method generates double-stranded 24 DNA fragments that are suitable for the direct use in NGS library construction, and allows to 25 eliminate the need of an additional step of reparation of DNA ends. 26 27 100 and four NGS libraries from four different samples of FTP-digested gDNA. NGS libraries were 101 generated using NEBNext Ultra II DNA Library Prep kit (New England Biolabs, Inc.) according to 102 the manufacturer's instruction. The conventional procedure for Fragmentase digested DNA 103 included: repair of DNA ends with "NEBNext Ultra II End Prep Enzyme Mix", addition of adapters 104 to the DNA fragments by "NEBNext Ultra II Ligation Master Mix" and amplification of the 105 adaptor-ligated DNA fragments with "NEBNext Ultra II Q5 Master Mix". Input amount of each 106 DNA sample was 200 ng. The library indexing and amplification were performed for 5 PCR cycles 107 as described in the kit's manual. 5 108 NGS libraries from FTP digested gDNA were constructed by NEBNext Ultra II DNA Library 109 Prep Kit procedure, but with the exception of DNA end reparation stage. 110 After the amplification stage, all libraries were quantified with Quant-iT PicoGreen dsDNA 111 Assay Kit (Molecular Probes, Inc., Eugene, OR, USA), pooled (500 ng of each) and purified with 112 AMPure XP beads. 113 114Next Generation Sequencing (NGS) and bioinformatic analysis 115 The pooled libraries were sequenced on the Illumina MiSeq Instrument (Illumina, California, 116 USA) with a 300 cycles MiSeq Sequencing Kit v2, paired-end mode, resulting in 12×10 6 reads. 117 Each of the reads was ~150 nt long. The FASTQ files generated on the instrument were uploaded to 118 NCBI SRArchive under project ID: PRJNA509202. 119 The FASTQ files were quality controlled using FASTQC v0.11.4 (Babraham bioinformatics, 120 Cambridge, UK). PHRED scores were calculated by FASTQC v0.11.4. Adapters were trimmed 121 with FLEXBAR v.2.5 [6]. Filtered reads with a minimum length of 30 bp were subsequently 122 aligned to the E.coli BL21(DE3) genome (NCBI Reference Sequence: NC_012971.2 ) using 123 BOWTIE2 software v2.3.4 [7]. Random samples of reads were generated using Seqtk software 124 (https://github.com/lh3/seqtk). De novo assembly of contigs was carried out with SPAdes tool 125 v3.10.1 130 We compared two enzymatic methods of dsDNA fragmentation for NGS library construction: 131 digestion with Fragmentase from New England Biolabs and Fragmentation Through Polymerization 132 (FTP). FTP method consists of two consequent enzymatic reactions: random DNA nicking and 133 elongation in a strand-displacement manner of the 3'-ends of nicked DNA. As a result, a number of
Nucleic acid fragments found in blood circulation originate mostly from dying cells and carry signs pointing to specific features of the parental cell types. Deciphering these clues may be transformative for numerous research and clinical applications but strongly depends on the development and implementation of robust analytical methods. Remarkable progress has been achieved in the reliable detection of sequence alterations in cell-free DNA while decoding epigenetic information from methylation and fragmentation patterns requires more sophisticated approaches. This review discusses the currently available strategies for detecting and analyzing the epigenetic marks in the liquid biopsies.
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